The study of tryptophol containing emulgel on fungal reduction and skin irritation

Tryptophol (TOH), a fungal quorum-sensing molecule, that possesses anti-fungal activities for controlling the growth of human pathogenic fungi. In the present study, we developed TOH-containing emulgel formulations and examined the antifungal activities and potential use as topical treatments on the skin. The results showed that TOH-containing emulgel at 1000 μM has excellent physical characteristics as homogenous, stability, and inhibits the growth of 30 species of human pathogenic fungi in vitro. TOH-containing emulgel did not cause skin irritation in mouse model of irritation and in healthy human volunteers. Moreover, an increase in skin hydration and a decrease in trans-epidermal water loss (TEWL) were observed after TOH-containing emulgel treatment on human skin. Our findings indicated that TOH-containing emulgel can be utilize as an antifungal agent for topical treatment against fungal infections on the skin.

Fungi can communicate to their extracellular environments by producing and releasing their small diffusible chemical signaling molecules known as fungal quorum-sensing molecules (QSM) 1 .Fungal QSMs have been shown to have a variety of biological activities such as cell-to-cell communication, morphogenesis, biofilm formation, and virulence, which ultimately controls the growth of another fungi 2 .To date, they are 4 main groups of QSMs including farnesol (FOH), tyrosol (TYR), phenylethanol (PE), and tryptophol (TOH) 3 .
Tryptophol (C 10 H 11 NO, TOH; molecular weight, 161.20) is a QSM that can be isolated from yeasts such as Candida albicans and Saccharomyces cerevisiae 4 .Our previous studies have demonstrated that TOH can inhibit the growth of Candida albicans 5 and Scedosporium apiospermum 4 .Thus, TOH has a high potential to be developed as an innovative product containing active antifungal agent for the treatment of fungal infections.
To develop a product containing TOH, vehicles that are used to incorporate TOH for dermal delivery are greatly concerns 6 .Repeated topical application of TOH may adversely cause skin irritation and contact dermatitis 7 .Emulsion-based formulations are currently the chosen formulation for topical delivery of active agents, which show good interaction on the skin 8 .
The aim of the study was to investigate the antifungal effects TOH-containing emulgel in vitro and skin irritation in vivo as well as on healthy human volunteers.Moreover, the cosmeceutical benefits of TOH-containing emulgel was also investigated.

Formulation and evaluation of TOH-containing emulgel
With the aim to develop the TOH-containing emulgel from emulgel base recipe, the emulgel prototype products containing 100 μM and 1000 μM of TOH were successfully developed.The physical and chemical properties of TOH-containing emulgel were evaluated previously 9 .Moreover, an emulgel containing 1000 μM of voriconazole (VOZ) was also produced.The parameters of emulgel base, TOH-and VOZ-containing emulgel were presented in Table 1.The manufactured 100 μM and 1000 μM of TOH-and 1000 μM of VOZ-containing emulgel appeared as white, translucent, homogenous, and non-greasy.However, high concentrations of active compounds (TOH and VOZ) caused an increase in viscosity and reduced the spread ability of the products (Table 1).There was no phase separation found both all manufactured emulgel formulations.There were no color changes or physical instability involving phase separation or inconsistency increases.Nevertheless, the pH of the manufactured emulgel formulations were found to be in the acceptable range of 4.5-5.5.

Evaluation of TOH-containing emulgel on mouse skin irritation
Single irritation test on mouse skin was used examined an acute irritant contact dermatitis (AICD) of 1000 μM TOH-containing emulgel.After 24 h post-topical application, croton oil and 5% Sodium Lauryl Sulfate (SLS) were able to induce mild to moderate edema with loss of skin creases, whereas, emulgel cream base, commercial shielding cream, 1000 μM VOZ-containing emulgel and 1000 μM TOH-containing emulgel did not induce any skin changes as determined by visual assessment.The notable histological features of acute irritant contact dermatitis were spongiosis, accumulation of fluid in the epidermis, and inflammatory cells infiltrations in epidermis, dermis, and subcutaneous tissues in the group treated with croton oil (Fig. 2d) and lessened in the group treated with 5% SLS (Fig. 2e).No histological characteristics of AICD were found in the group treated with emulgel cream base (Fig. 2a), commercial shielding cream (Fig. 2b), 1000 μM VOZ-containing emulgel (Fig. 2c), and 1000 μM TOH-containing emulgel (Fig. 2f).
Cumulative irritation test on mouse skin was used examined a chronic irritant contact dermatitis (CICD) of 1000 μM TOH-containing emulgel.Severe epidermal hyperplasia, spongiosis, inflammatory cells infiltrations, and dilatation of dermal vessels were found in the group treated with croton oil after 7 days (Fig. 3d) and 14 days of application (Fig. 4d) and lessened in the group treated with 5% SLS after 7 days (Fig. 3e) and 14 days of application (Fig. 4e).No histological characteristics of CICD were found in the group treated with emulgel cream base (Figs.3a and 4a), commercial shielding cream (Figs.3b and 4b), 1000 μM VOZ-containing emulgel (Figs.3c and 4c), and 1000 μM TOH-containing emulgel (Figs.3f and 4f) after 7 days and 14 days of application, respectively.Therefore, the present study suggested that 1000 μM TOH-containing emulgel did not induce neither acute or chronic irritation in vivo.

Evaluation of TOH-containing emulgel on human skin irritation
Skin irritation test was determined in thirty healthy volunteers after topical treatment of 1000 μM TOH-containing emulgel.After 24 h post-topical application, no visual characteristics of skin irritation or dermatitis observed in the group treated with commercial shielding cream (Fig. 5a), 1000 μM TOH-containing emulgel (Fig. 5b), 1000 μM VOZ-containing emulgel (Fig. 5c), and emulgel cream base (Fig. 5d).Moreover, no skin irritation was observed after 72 h post-topical application in all treatments (Fig. 5).Therefore, the present study suggested that 1000 μM TOH-containing emulgel treatment did not cause skin irritation in human volunteers.

Evaluation of TOH-containing emulgel on human skin physiological properties
Hydration values measured on the skin of volunteers treated with 1000 μM TOH-containing emulgel are significantly higher than emulgel base and distilled water treated areas (Fig. 6a).Skin hydration was significantly Table 2. Colony diameters of tryptophol (TOH)-containing emulgels treated fungal pathogens after incubation at 37 °C for 14 days.TOH Tryptophol, VOZ voriconazole.a-c Indicates statistically significant P<0.05 in each experimental condition by two-way ANOVA.increased after treated with 1000 μM TOH-containing emulgel (Fig. 6b).Moreover, treatment of 1000 μM TOH-containing emulgel significantly reduced transepidermal water loss (TEWL) than other products (Fig. 6c).Therefore, the present study suggested that 1000 μM TOH-containing emulgel treatment enhances other cosmeceutical benefits to the human skin.

Discussion
Superficial mycosis is caused by pathogenic fungi that affects superficial layer of the skin 14 .Several species of human pathogenic fungi can infect deeper than the superficial layer of the skin causing cutaneous mycosis 15 .These skin mycoses are common worldwide, which in some cases, causing cosmetically poor appearance with severe inflammation 16 .Antifungal drug such as voriconazole (VOZ), a triazole compound, is the drug-of-choice antifungal agent against fungal infections 17 .However, fungal infections that resistant to triazoles such as VOZ has become a concern with increases cases over the past decade 18 .Thus, the present study was conducted and developed to evaluate the effectiveness of alternative topical treatment to treat various pathogenic fungi as a tryptophol (TOH)-containing emulgel.
In general, TOH can be naturally found as a quorum sensing molecule (QSM) in seed plants, bacteria, and fungi 19 .TOH affects fungi morphogenesis, which provides a potential use as an antifungal agent various fungal pathogen species 20 .We have previously demonstrated that in vitro treatment of TOH suppresses the pathogenicity of C. albicans.In addition, treatment of TOH was found to induce C. albicans apoptosis via the transcriptional upregulation of caspase recruitment domain-containing protein (CARD)-9 and Noxa and the downregulation of Bcl-2 5 .We also previously demonstrated that treatment of TOH at 100 μM reduces the germ tube length and biofilm formation of S. apiospermum in vitro.Moreover, TOH-containing emulgel was developed at 100 μM, which reduced S. apiospermum-induced eumycetoma formation in vivo 9 .Also the antifungal activity of TOH both in vitro and in vivo have been extensively studied, whether antifungal activity and safety of TOH-containing emulgel for clinical use remains to be elucidated.
In the present study, we demonstrated that TOH at 100 μM and 1000 μM was successfully incorporated into emulgel base containing SEPIGEL 305 9 .Moreover, TOH-containing emulgel in the present study exhibited excellent in cosmeceutical levels including homogeneity, consistency, and stability with pH range of 5.5, which compromised with the pH of the skin 21 .Interestingly, we further demonstrated that 1000 μM TOH-containing emulgel exhibited excellent in antifungal activity against 30 pathogenic fungi including azole-resistant strains, hyaline and dematiaceous molds, dimorphic fungi, and yeasts than 1000 μM VOZ-containing emulgel in vitro.Thus, our study suggested that the manufactured TOH-containing emulgel at 1000 μM was optimal for topical use in vivo with antifungal activity against wide ranges of human cutaneous pathogenic fungi.
In this report, we demonstrated that TOH caused less cytotoxic to human foreskin fibroblast (HFF-1) cells than VOZ in vitro suggesting that TOH is safe to use as topical treatment in vivo.We further determined the safety use of TOH-containing emulgel in human volunteers by determining both acute irritant contact dermatitis (AICD) and chronic irritant contact dermatitis (CICD) caused by 1000 μM TOH-containing emulgel prior to commercialize the product.From the present study, our results suggested that either single or repeated exposure of 1000 μM TOH-containing emulgel does not cause skin irritation or any histological changes, which suitable for long term use 22 .
No skin irritation was observed in human volunteers after 1000 μM TOH-containing emulgel treatments.Moreover, skin hydration and less water loss were gained after 1000 μM TOH-containing emulgel treatments.Therefore, our present study showed that 1000 μM TOH-containing emulgel is suitable to be used as an alternative antifungal product for topical treatment.Hence, it is possible to infer that the 1000 μM TOH-containing emulgel was safe for topical application on human skin without skin adverse effects.Nevertheless, the antifungal activity of 1000 μM TOH-containing emulgel need to be evaluated further in patients with cutaneous fungal infections, which we plan to investigate in the near future.

Conclusions
The study confirmed positive effects of 1000 μM TOH-containing emulgel that exhibits antifungal activities and cosmeceutical benefits without causing skin irritation to human.The potential use of 1000 μM TOH-containing emulgel in patients with skin mycoses may be investigated in the future.Further, this study showed an innovative approach to utilize TOH-containing emulgel as a topical application for treating fungal infections as being in accordance with the growing use of emulgels in cosmeceutical and pharmacological products.

Preparation of TOH-containing emulgel formulation
The emulgel formulations were prepared with either tryptophol or voriconazole according to Kitisin et al. 9 .Optimization of all the emulgel formulations were performed by evaluating the effect of different concentrations of excipients based on visual changes in appearance, homogeneity, consistency, viscosity, stability, and any change in the physical characteristics prior to manufacturing the final formulations.The compositions of all the optimized emulgel formulae are provided in Table 3.

Manufacturing process for emulgel
The emulgel base was manufactured with a constant mechanical stirring of the gel-based SEPIGEL 305 ™ .TOH at 100 µM and 1000 µM or VOZ at 1000 µM were accurately prepared and added to the emulgel base and vigorously mixed until a smooth emulgel was formed 9 .

Visual evaluation
The visual appearance of manufactured emulgel formulations were examined for their physical appearances including color, homogeneity, consistency, and phase separation 23 .

Measurement of viscosity and pH
The viscosity of manufactured emulgel formulations was evaluated at room temperature using a Brookfield viscometer (Brookfield, Model programmable DV2, USA) with a volume sample of 0.5 g 9 .The formulated emulgel samples were left for equilibrium (30 min) at 25 °C.Measurements with the spindle were determined at 100 rpm for 10 min.The viscosity reading of each sample was recorded.Moreover, the pH of all manufactured emulgel formulations was measured by a digital pH meter (Thermo Fisher Scientific, Waltham, MA, USA) 23 .
Prior measurement, pH meter was calibrated using standard pH buffers at 4, 7, and 10.Then, the pH for all the emulgel formulations was recorded.The pH meter probe was washed after each measurement with DI water and 70% (v/v) EtOH.Data were analyzed in tripiclate.

Measurement of spreadability
The spreadability of manufactured emulgel formulations was investigated to determine the competence of each emulgel formulation, which is able to spread evenly after smeared on the affected skin 24 .To measure the spreadability of emulgel samples, 1 g of each formulated emulgel was placed onto the ground fixed glass plate and covered with another glass plate and placed on a wooden block.A definite load of about 20 g was then placed on the top of the slides for 1 min to provid the basis for the slip and drag characteristics of the emulgel.The time in seconds required to separate the two slides was recorded and analyzed in triplicate.The spreadability was calculated using the following equation: S = m * l/t; where S stands for spreadability, m stands for weight tied on upper slide; l is the length of glass slides; t, the time (s) requires to separate the slides completely 25 .

Measurement of stability
Physical stability studies were investigated for all the formulations using using centrifugation and temperature cycle tests 9 .For centrifugation test, approximately 10 g of sample was centrifuged at 9000 rpm for 30 min.In addition, 2 g of each prepared emulgel samples was subject to a temperature cycle test by storing in 24 h/a cycle at − 4 °C (8 h) and 40 °C (16 h) for 10 cycles.After cooling-heating and freeze-thaw, the emulgel samples were evaluated for stability changes.All measurements were performed in triplicate.Physical stability of these emulgels formulations was evaluated at the end of these tests.

In-vitro antifungal activity studies
In-vitro antifungal activity studies of manufactured emulgel formulations were evaluated using the agar spot assay according to Horváth et al. 27 for yeasts and according to Muangkaew et al. 28 for filamentous fungi with some modifications.Briftly, ten µl of each fungal conidia at concentration about about 5 × 10 6 conidia/ml were mixed with 10 µl of each manufactured emulgel formulations (TOH at 100 or 1000μM or VOZ at 1000 μM) and spotted on the surface of SDA plates.Then, the plates were incubated at 37 ± 1 °C for 14 days.The colony diameter (mm) were measured in millimeters using a vernier caliper 4,9 .Uninoculated SDA plate was used as a negative control.The experiment was performed in triplicate.

Cell culture, viability, and cytotoxicity
Human foreskin fibroblasts (HFF-1) cell line (ATCC #SCRC-1041™) was used to investigate cell viability and cytotoxicity after TOH treatments.HFF-1 cells were cultured in RPMI-1640 supplemented with 10% FBS and 100 U/ml of Pen-strep and incubated under humidified conditions with 5% CO 2 at 37 °C.Measurements of cell viability and cytoxicity after TOH treatments were carried using MTT 9 , CytoPainter MitoGreen, LDH 9 , and dual AO/EB staining assays.As previously described, HFF-1 were seeded at a concentration of 1 × 10 5 cells/ml in a 96 well plate and after 24 h were treated with 100 μl of TOH at 100 or 1000μM or VOZ at 1000 μM or with absolute EtOH and RPMI 1640 at 1% (v/v) as diluent controls.Untreated HFF-1 cells were used as negative controls (100% viable).After incubation for 24 h, 10 μl of 0.5 μg/μl MTT solution or 50 μl of CytoTox 96 ® reagent were added and measured spectrophotometrically at 540 nm or 490 nm, respectively (Sunrise Tecan, Grödig, Austria).To label cell viability and toxicity, treated HFF-1 cells were stained with 100 μl of MitoGreen indicator or 25 μl of 100 μg/μl AO/EB solution, respectively.Cellular morphology and apoptotic cell numbers were examined by a Zeiss Axio Imager fluorescence microscope (ZEISS, Germany).All of the experiments were performed in triplicate.

In vivo evaluation of the emulgel formulations on mouse skin irritation
Female BALB/c mice were purchased from Nomura Siam International Co, Ltd. (Thailand) and used to evaluate skin irritation of manufactured emulgel formulations.Each group included 5 animals (aged 6-8-week-old, body weight: 20-25 g) The skin irritation mouse model was produced and validated as described previously with some modification 22 .Briefly, all mice were topically treated with various concentrations of manufactured emulgel formulations (including emulgel base, 1000 µM of TOH or VOZ containing emulgels) that do not cause necrosis of the epidermis.Five percentage of SLS and croton oil were used as irritants.Commercial shielding cream was applied as controls.For single irritation, 15 μl of the irritants were put on the 8 mm Finn Chambers on Scanpor tape (Epitest Ltd., Finland), which were then applied on the dorsal skin of the animals.Polyurethane film (3M™ Tegaderm™, NY, USA) was used to fix them and Finn Chambers were then removed after 24 h postapplication.For cumulative irritation, the procedure was repeated once a day for 1 and 2 weeks.After removing the Finn Chambers, mice were sacrificed, and skin samples were collected.The specimens were fixed in 10% buffered formalin and processed for haematoxylin and eosin (H&E) stain.All skin sections were examined with a light microscope (BH-2, Olympus, Japan).All animal experiments were conducted in accordance with the Animals for Scientific Purposes Act, B.E. 2558 (A.D. 2015), Thailand.Experiments on mice were approved by Institutional Animal Care and Use Committee (IACUC) of the University of Phayao, Thailand (approval number: UP-AE64-01-04-017).

In vivo evaluation of the emulgel formulations on human skin irritation
To evaluate the sensitization potential of manufactured emulgel formulations toward human skin, a skin irritation test was performed by 30 healthy volunteers 29 .A single irritation test of the manufactured emulgel formulations (emulgel base, 1000 µM of TOH or VOZ containing emulgels) were put on the 8 mm Finn Chambers on the back of arm skin of the volunteers and fixed with polyurethane film.Commercial shielding cream was applied as controls.Finn Chambers were then removed after 24 h post-application.Treated areas of volunteer skin were observed immediately and 72 h after removing Finn Chambers.All in vivo studies with healthy volunteers are complied with the 2013 Declaration of Helsinki and was approved Ethics Committee (EC) at the Faculty of Tropical Medicine, Mahidol University, Thailand (approval number: MUTM 2022-036-01).Written informed consent was obtained from all the participants.

In vivo evaluation of the emulgel formulations on human skin hydration, trans-epidermal water loss (TEWL), and skin color
To evaluate the effects of manufactured emulgel formulations on human skin physiological functions including skin hydration and trans-epidermal water loss (TEWL), treated skins of 30 healthy volunteers with manufactured emulgel formulations were investigated using DermaLab ® with a hydration probe and a TEWL probe (Cortex Technology, Hadsund, Denmark) 30 .After manufactured emulgel treatments, measurements of the skin hydration and TEWL were performed in 2 × 2 cm squares on the middle forearms, next to each other, with an interval of 5 s between each measurement.Three measurements were taken in each square.

Table 1 .
Physical properties of different optimized emulgel formulations.Values are expressed as mean ± standard deviation (SD).TOH Tryptophol, VOZ voriconazole.a-d Indicated P < 0.05 compared between experimental conditions by one-way ANOVA followed by Tukey's multiple comparison test.